Dry biochemical assay plate and method for making the same

ABSTRACT

An assay plate for detecting the presence of a mobile reactant that binds to a immobilized reactant and the methods of making and using the same. An assay plate according to the present invention includes a substrate and at least one dried aliquot of the immobilized reactant, the immobilized reactant being bound to the surface of the substrate. The immobilized reactant binds the mobile reactant when a solution containing the mobile reactant is brought into contact with the immobilized reactant. The mobile and immobilized reactants may be any pair of biological compounds that have a specific affinity for one another. For example the reactants may be nucleic acids or antibody-antigen pairs. The preferred embodiment of an assay plate according to the present invention includes a plurality of assay spots, each spot having a different immobilized reactant or concentration thereof. The preferred method for fabricating an assay plate according to the present invention includes the steps of binding the immobilized reactant to the substrate, washing the substrate to remove any immobilized reactant that is not bound to the substrate and then drying the substrate. The dried assay plates are preferably stored in a water-proof container until used. An assay utilizing an assay plate according to the present invention is carried out by bringing a solution containing the mobile reactant into contact with the dried aliquot or aliquots on the assay plate. The assay plate is then washed to removed unbound material and the amount of mobile reactant bound to the washed assay plate determined. In the preferred embodiment of the present invention, the washed assay plate is dried prior to measuring the amount of mobile reactant bound to the washed assay plate.

This is a continuation of application Ser. No. 09/337,710 filed on Jun.21, 1999, now U.S. Pat. No. 6,255,053, which is a continuation of Ser.No. 08/412,498 filed on Mar. 28, 1995, now U.S. Pat. No. 5,922,534, fromboth of which priority is claimed and both of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to biochemical assays, and moreparticularly, to assays in which the presence of a target reactant isdetermined by measuring the amount of material that is bound to animmobilized reactant.

BACKGROUND OF THE INVENTION

Reactions between biological molecules exhibit an extremely high degreeof specificity. It is this specificity that provides a living cell withthe ability to carry out thousands of chemical reactions simultaneouslyin the same “vessel”. In general, this specificity arises from the “fit”between two molecules having very complex surface topologies. Forexample, an antibody binds a molecule displaying an antigen on itssurface because the antibody contains a pocket whose shape is thecomplement of a protruding area on the antigen. This type of specificbinding between two molecules forms the basis of numerous biologicalassays.

For example, nucleic acids are linear polymers in which the linkedmonomers are chosen from a class of 4 possible sub-units. In addition tobeing capable of being linked together to form the polymers in question,each unit has a complementary sub-unit to which it can bindelectrostatically. In the case of DNA, the polymers are constructed fromfour bases that are usually denoted by A, T, G, and C. The bases A and Tare complementary to one another, and the bases G and C arecomplementary to one another. Consider two polymers that are alignedwith one another. If the sequences in the polymers are such that an A inone chain is always matched to a T in the other chain and a C in onechain is always matched to a G in the other chain, then the two chainswill be bound together by the electrostatic forces. Hence, animmobilized chain can be used to bind the complementary chain. Thisobservation forms the basis of tests that detect the presence of DNA orRNA that is complementary to a known DNA or RNA chain. Such detectionforms the basis of a number of medical and/or diagnostic tests.

The methods by which the binding of the mobile reactant to theimmobilized component of the system is measured varies with theparticular reactants. However, a significant fraction of all of thetests involve the measurement of a fluorescent dye that is associatedwith either the bound or mobile reactant. The dye may be attached to thereactant from the beginning of the process or it may be added throughvarious chemical steps after the mobile and immobilized reactants havebeen brought into contact with one another.

Systems for medical diagnosis often involve a bank of tests in whicheach test involves the measurement of the binding of one mobilecomponent to a corresponding immobilized component. To provideinexpensive test kits, systems involving a matrix of immobilized spotshave been suggested. Each spot includes the immobilized component of atwo component test such as described above. The fluid to be tested istypically brought into contact with the matrix. After chemicalprocessing, the amount of fluorescence associated with each of the spotsin the matrix is measured.

The matrix is typically constructed by dispensing small quantities ofthe immobilized component onto a substrate such as glass or filterpaper. In general, prior art assays utilizing such matrices require thatthe matrix remain wet from the point in the process at which thecomponents are dispensed through the detection of the fluorescence. Thisrequirement leads to a number of problems when these assay are appliedin medical diagnosis. First, the buffer solutions utilized in theprocessing may contain contaminants that have fluorescent emission bandssufficiently close to those of the fluorescent compound of interest thatthe stray fluorescence gives rise to errors in the assay. The amount ofinterference depends on the amount of buffer needed in the particularsystem. If the ratio of buffer solution to bound fluorescent compound ishigh, even a small degree of contamination of the buffer solution cangenerate unacceptable errors.

A second problem with wet assay plates relates to the transportation andstorage thereof. In medical diagnostic applications, it is anticipatedthat the assay plates will be prepared by a commercial supplier andshipped to the diagnostic laboratory. The plates would then be stored atthe laboratory until needed. The need to provide leak-proof packagingsignificantly increases the cost of these assay plates.

In addition, the short storage life of wet assay plates at roomtemperature places additional constraints on the storage andtransportation of these assay plates. The biological macromolecules onwhich these assays are based are easily attacked by various enzymes.These enzymes often appear as contaminants in the various buffersolutions used in preparing and storing the wet assay plates. As aresult, the assay plates must be refrigerated to increase their shelflife. The cost of refrigerated storage and transportation significantlyincreases the cost of assay systems based on wet assay plates.Furthermore, even with refrigerated storage, the useful shelf life ofwet assay plates results in significant increases in costs due to theneed to discard old assay plates before they are actually used.

A third problem with wet assay plates is the need to read the results ofthe assay shortly after the chemical processing of the plates. Thisrestricts the reading and interpretation of the results to thelaboratory that processed the patient samples with the plates. Whilethis restriction is not very significant in metropolitan settings, itcan be a significant problem in rural settings. In which the volume oftests is too low to justify the cost of the equipment and personnelneeded to read and interpret the assays. As a result, the patientsamples are typically sent to a central laboratory for reading andprocessing. The need for refrigerated transportation of the samples andthe inherent time delays in receiving the results of the tests make thissolution to the problem less than optimal.

Finally, prior art wet assay plate systems do not provide a means forarchiving the assay plates for later examination since the catabolicenzymes described above will destroy the underlying macromolecules evenif the assay plates are stored in a refrigerated environment. An assayplate that provided a cost effective archival storage mechanism whichwould allow the plate to be read again some time after its originalprocessing would be highly desirable both from a research and a legalpoint of view. Researchers often wish to examine samples from a largepopulation. The samples in question can often be taken from routineassays if archival storage of the routine samples is available.Unfortunately, present archival storage requires storage of the originalsamples at liquid nitrogen temperatures. The costs inherent in thisapproach limit the archiving of samples to special studies.

In legal settings, the ability to re-examine tests performed months oryears earlier would be of significant benefit in determining thevalidity of the earlier performed tests. There are many situations inwhich the validity of such tests determines the outcome in a legalproceeding.

Broadly, it is the object of the present invention to provide animproved assay plate system for performing assays based on the bindingof biological macromolecules.

It is a further object of the present invention to provide an assayplate system in which the assay plates do not need to be shipped orstored in a wet state.

It is a still further object of the present invention to provide anassay plate system in which the assay plates may be read and archived ina dry state.

These and other objects of the present invention will become apparent tothose skilled in the art from the following detailed description of theinvention and the accompanying drawings.

SUMMARY OF THE INVENTION

The present invention comprises an assay plate for detecting thepresence of a mobile reactant that binds to a immobilized reactant andthe methods of making and using the same. An assay plate according tothe present invention includes a substrate and at least one driedaliquot of the immobilized reactant, the immobilized reactant beingbound to the surface of the substrate. The immobilized reactant bindsthe mobile reactant when a solution containing the mobile reactant isbrought into contact with the immobilized reactant. The mobile andimmobilized reactants may be any pair of biological compounds that havea specific affinity for one another. For example the reactants may benucleic acids or antibody-antigen pairs. The preferred embodiment of anassay plate according to the present invention includes a plurality ofassay spots, each spot having a different immobilized reactant orconcentration thereof. The preferred method for fabricating an assayplate according to the present invention includes the steps of bindingthe immobilized reactant to the substrate, washing the substrate toremove any immobilized reactant that is not bound to the substrate andthen drying the substrate. The dried assay plates are preferably storedin a water-proof container until used. An assay utilizing an assay plateaccording to the present invention is carried out by bringing a solutioncontaining the mobile reactant into contact with the dried aliquot oraliquots on the assay plate. The assay plate is then washed to removedunbound material and the amount of mobile reactant bound to the washedassay plate determined. In the preferred embodiment of the presentinvention, the washed assay plate is dried prior to measuring the amountof mobile reactant bound to the washed assay plate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a side view of an assay plate according to the presentinvention.

FIG. 2 is a top view of the assay plate shown in FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be more easily understood with reference toFIGS. 1 and 2 which are side and top views of a matrix of test spots 12deposited on a substrate 10. The individual spots are typicallydispensed by a dispensing apparatus 14. Each test spot includes onemember of a pair of chemical structures that will bind to one another ifbrought into contact with one another. The chemical species included inthe test spot will be referred to as the immobilized species. The othermember of the pair will be referred to as the mobile species.

The immobilized species is typically deposited on substrate 10 in acarrier liquid. In principle, each spot includes a different immobilizedspecies or concentration thereof that will become attached to substrate10. The preferred method for providing the attachment will be discussedin more detail below. The presence of a mobile species is determined bymeasuring the amount of material bound to the corresponding test spotwhen a solution to be tested is brought into contact with the test spot.Test plates such as that shown in FIGS. 1 and 2 are designed to test fora plurality of mobile species simultaneously.

The manner in which the test spots are generated may be more easilyunderstood with reference to a test spot for detecting a DNA moleculehaving a specific nucleotide sequence. As noted above, a single strandedDNA molecule will bind to a second single stranded DNA molecule if thesecond DNA molecule has a sequence that is complementary to the firstDNA molecule. The amount of bound DNA may be measured photometrically bymeasuring the amount of dye present when the bound test spot is treatedwith a dye that binds to double stranded DNA molecules. Alternatively,the second DNA molecules can be labeled with a dye that can be measuredphotometrically.

The present invention is based on the experimental observations that theassay plates may be dried at two key points in the assay process. First,the assay plates may be dried after the immobilized species have beenbound to the assay plate. This allows the assay plates to be stored asdry plates. The dry plates may then be used in assays to detect themobile species by re-hydrating the plates and exposing the plates to themobile species utilizing the same conventional wet chemistry that isused in the analogous assay system utilizing conventional wet assayplates. After the reactants have bound to the immobilized species andthe reaction is complete, the assays may be read by conventional meansor as described below. The dried assay plates are found experimentallyto have significantly longer shelf-lives than the conventional wetplates, since the degradative process that normally limit the life timeof wet plates proceed much slower in the absence of water.

While the assay plates may be read in their final wet state, in thepreferred embodiment of the present invention several wash steps areperformed with the appropriate buffers to remove any non-specificbinding component and the assay plates are once again dried. The driedplates may then be read in a fluorescent detection system withoutinterference from contaminants in the buffers. Alternatively, the driedassay plates may be stored for later reading. This alternative allowsthe plates to be read at a remote location and/or archivally stored.

Having provided the above overview of the present invention, the mannerin which the assay plates are prepared will now be described in moredetail. For the purposes of this discussion, it will be assumed that theassay plates are constructed on glass substrates, specifically, fusedsilica slides and that the immobilized species is a oligonucleotide,either DNA or RNA. However, procedures for binding other immobilizedspecies will be apparent to those skilled in the art from the followingdiscussion. It should also be noted that the basic chemistry for bindingthe immobilized species to a glass surface is well known to the art. Thefollowing discussion is provided to provide the reader with anappreciation of the procedure.

The process starts with the cleaning of the slides. In the preferredembodiment of the present invention, the slides are sonicated indetergent for 15 minutes. The preferred detergent solution is 5% RBS-35(Pierce Chemicals)/95% ethanol. The slides are then rinsed for 10minutes in double distilled water. The slides are then subjected to aconcentrated nitric acid wash for 15 minutes followed by a seconddistilled water rinse. The cleaned slides are dried in two steps. First,the slides are dried with N₂. The slides are then placed in an oven at110° C. for 15 minutes and dried under vacuum.

The cleaned slides are then reacted with amino propyl triethoxy silane(APTES) to provide binding sites for attaching the immobilized species.The surface of the slide is coated with a 1% APTES solution in 95%ethanol. The slides are incubated at room temperature for 45 minutes ina covered petri dish. The slides are then washed in 95% ethanol anddried in N₂ at 110° C. for 15 minutes. The drying is preferably carriedout by the two step procedure discussed above. The coated slides may bestored in aluminum foil or in a N₂ filled chamber.

The immobilized species is then linked to the coated surface via Bissuccinimydl suberate-homobifunctional NHS-ester (BS³). This procedure isperformed in two steps. First, the immobilized species is linked to theBS³ linker. The linked immobilized species is then attached to the APTEScoated surface. The linkage reaction is carried out by mixing 20 μl ofBS³ to 55 nmoles of the immobilized species in a TES buffer andincubating the mixture for 15 minutes at room temperature. The linkedimmobilized species are then purified on a Nap 25 column (available fromPharmacia).

The linked immobilized species dissolved in a TES buffer at pH 8.0 arethen placed on the APTES coated surface as a plurality of “spots” on thesurface, each spot containing a different immobilized species orconcentration thereof. The slides are incubated for 1 hour at roomtemperature in a moist chamber. Any excess fluid is removed byaspiration. The slides are then washed in TES buffer at pH 8.0. Theslides are then dried in N₂. In the preferred embodiment of the presentinvention, the slides are packaged in moisture proof packaging forstorage.

The above described attachment scheme utilizes the amino group of theimmobilized species as the “hook” for attaching the immobilized speciesto the slide. Hence, it will be apparent to those skilled in the artthat proteins may also be attached via this mechanism. In particular, itwill be apparent from the above discussion that assay plates based onimmobilized antibodies or antigens may also be prepared utilizing thisprocedure.

As noted above, the dried slides may be used in place of conventionalwet slides at the time the assay for the mobile species is to beperformed. A solution containing the mobile species is brought intocontact with the surface of dried slide under conditions that permit thebinding of the mobile species to the immobilized species. After anappropriate incubation period, the slides are washed to remove anyunbound immobilized species. The amount of material bound to the washedslides is then determined by applying a dye that binds to either thebound immobilized-mobile species complex or to the mobile species. Forexample, in applications in which the mobile and immobile species areboth nucleic acids, acridine dyes that insert themselves between the twobound strands of nucleic acid may be used. In the case ofantibody-antigen assays, the dye system detects bound antibodies byattaching to the general class of antibodies. This later case involves anumber of steps that are conventional in the art, and hence, will not bediscussed in more detail here.

It has also been observed experimentally that the slides with the bounddyes may be washed and dried without substantially interfering with themeasurement of the bound dye. In fact, dry slides often have lowerbackground readings than slides read in the conventional manner, sinceany contamination from fluorescent compounds in the buffer solutionsnormally used in the wet process are substantially reduced. In thepreferred embodiment of the present invention, the slides are washed asdescribed above and dried in N₂.

The dried slides may be stored for months or even years and read aplurality of times if necessary. This ability to store the final slidesallows slides prepared at a remote location to be sent to a centralreading location. In addition, the accuracy of tests may be verified ata later time, thus reducing false positives resulting from errors inreading the original reading of the slides. The ability to verify a testresult is particularly useful in legal settings in which the validity oftest results is often questioned month or even years after the testswere performed.

Various modifications to the present invention will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Accordingly, the present invention is to be limited solely bythe scope of the following claims.

1. An assay plate comprising: a glass substrate comprising a pluralityof dried spots, each spot comprising a different concentration of animmobilized oligonucleotide, wherein oligonucleotides at one or morespots have bound thereto a complementary molecule and wherein the assayplate is in a waterproof storage container.
 2. The assay plate of claim1, wherein the complementary molecule comprises a label.
 3. The assayplate of claim 2, wherein the label is a fluorescent label.
 4. The assayplate of claim 1, wherein the oligonucleotides comprise DNA.
 5. An assayplate comprising: a glass substrate comprising a plurality of spots,each spot comprising a different concentration of an immobilizedoligonucleotide, wherein oligonucleotides at one or more spots havebound thereto a complementary molecule and wherein the assay plate isdry.
 6. The assay plate of claim 5, wherein the assay plate is in awaterproof storage container.
 7. The assay plate of claim 5, wherein thecomplementary molecule comprises a label.
 8. The assay plate of claim 7,wherein the label is a fluorescent label.
 9. The assay plate of claim 5,wherein the oligonucleotides comprise DNA.